Method of and apparatus for investigating tissue histology

ABSTRACT

Apparatus for monitoring the presence of one or more chromophores in a tissue sample, comprises a light source for projecting light to illuminate an area of such tissue sample, a photo-receptor for receiving light remitted by the illuminated area of tissue, and spectroscopic analyser means for monitoring the remitted light, a comparator having means for comparing variations in the intensity and spectral characteristics of the remitted light with respect to the intensity and spectral characteristics of the projected light at different wavelengths and with a record of the intensity and spectral characteristics of light remitted by a reference sample of such tissue and means for emitting a control signal in response to any such variations. Methods of analyzing tissue histology, especially skin histology, are discussed, and a mathematical model is proposed for the analysis and comparison of the remitted light with a reference sample.

This invention relates to a method of and apparatus for theinvestigation of tissue histology. The invention has particularreference to the investigation of chromophores within layers close tothe surface of such tissue, and while the invention may be applied inthe investigation of laboratory tissue specimens, whether obtained froma biopsy or necropsy, it was developed with the particular intention ofenabling in vivo observation of a subject without the need for anysurgical intervention which might expose the subject, or indeed thesurgeon, to the risk of infection. The invention is thus applicableespecially to the investigation of epithelial tissue, such as the skinand linings of the respiratory and digestive tracts and other surfacesto which visual access may be had, such as the retina, without removingthe tissue being investigated from the body of the subject. Theinvention is particularly applicable to the non-invasive investigationof tissue histology.

In order to appreciate the presence of abnormalities in the tissue beingexamined it is first necessary to have an appreciation of the structureof normal tissue of that type.

Though the invention may be adapted for the investigation of otheranimal tissue, it was originally developed with particular regard to theinvestigation of conditions whose symptoms include abnormalities in thehuman skin, and it is in that context that it will be particularlyexplained.

Thus, in order to appreciate the presence of abnormalities in the skinit is first necessary to have an appreciation of the structure of normalskin.

The presence and extent, including depth and concentration, ofchromophores within epithelial tissue such as the skin is considered tobe an important indicator of a variety of ailments and other conditions.The invention is considered to be potentially useful for the preliminaryscreening of patients to identify those who should be referred to anappropriate clinician for diagnosis and further to assist the clinicianin diagnosis and in some embodiments to indicate whether a giventreatment would be of value to the patient, and for other purposes.

The skin is divided into two main layers, the epidermis and the dermis,each of which is itself divided into several sub-layers. Starting fromthe deepest layer, the subcutaneous layer is overlain by a reticularlayer of the dermis which is composed of coarse and dense interlacingbundles of collagen fibers (“type 1 collagen”) which are intermingledwith reticular fibers and elastic fibers. Over this is the papillarydermal layer which is also composed of collagen fibers but these aremuch finer than those of the reticular layer in that they are notbundled together. The collagen in the papillary dermis is mainly “type 3collagen”, and it constitutes connective tissue joining the epidermisand the reticular layer of the dermis. The dermis is also rich in bloodvessels. The papillary dermis is located immediately beneath theepidermis and is separated from it by the basal lamina. Thedermo-epidermal junction is highly irregular in profile due to dermalpapillae projecting up from the dermis between rete ridges or pegsprojecting down from the epidermis. It is the presence of these reteridges or pegs and papillae which gives the skin elasticity, and theirinteraction also provides an anchor for the epidermis. Epithelium cellsmultiply continuously in a germinative layer, just above the basallamina, to replace cells lost from the surface of the epidermis. Thegerminative layer, which is fed by blood vessels leading through thedermis, also contains melanocytes for the production of melanin. Theepithelium cells from the germinative layer move upwards into the layerabove, the spinous layer, and thence into the granular layer where thecells contain granules which are involved in the formation of keratin.It is in this granular layer that the cells of the epidermis die. Abovethe granular layer, is a clear and translucent layer and above that isthe outermost layer, the cornified layer. This is composed of clear deadscale-like skin which is progressively lost from the surface byexfoliation.

Historically, dermatological investigations have taken place by biopsy,that is by surgical removal of samples of skin tissue followed bymicroscopic examination of thin sections of the skin tissue usuallyviewed at right angles to the skin surface. The information obtained islimited in area to the thin section, unless a number of sections isexamined. Each section requires to be cut, stained and mounted onto amicroscope slide, and they are therefore time consuming to pre pare.Further the technique is invasive, and there may be a consequent risk ofinfection either at the biopsy site or from the biopsied material, orboth, unless stringent precautions are taken.

In normal circumstances, the healthy epidermis is translucent andtransmits light diffusely; a proportion of incident light will beabsorbed in the epidermis, depending in part on the amount of melaninpresent in the epidermis, and a proportion will be transmitted throughto the dermis. Because the papillary dermis largely consists of type 3collagen, that is, a very fine network of collagen fibers (as low as 2μm in diameter), light passing through the papillary dermis will besubject to Rayleigh scattering. A proportion of the incident light willbe scattered inwards and a proportion will be back-scattered, and someof this scattered light will be remitted back through the epidermis. Inthe reticular dermis the fibers are of type 1 collagen, that is, theyare clumped or bundled together, and they are largely parallel to theskin surface: thus they are too coarse to give rise to Rayleighscattering, and light penetrating to the reticular dermis will continueuntil absorbed or deflected by some discontinuity.

Thus light remitted by the epidermis will have its spectralcharacteristics altered by the effects of melanin, blood and otherchromophores in the skin. The mean thickness of the papillary dermis canvary quite considerably as between one part of the body and another, forexample, and in particular, the height and population density of dermalpapillae tends to increase according to the stress to which a particulararea of skin is habitually subjected. Thus, the thickness of thepapillary dermis over a joint will tend to be greater than that over arelatively non-stressed region such as the lower back. These variations,and variations between different subjects will have a marked effect onthe skin color, but we have found that it is possible to construct amathematical model which allows corrections to be made for this effect.When so corrected it is notable that the color of normal healthy humanskin lies in a well defined surface area within a particular colorspace, for example the CIE LMS color space. That surface areaencompasses all colors of normal healthy human skin irrespective of theamount of melanin within the skin and thus irrespective of race ordegree of tanning. This approach allows parameters relating tochromophores within the skin to be measured in a more accurate andrepeatable way through optical means than was permitted by previouslyexisting techniques.

According to the present invention, there is provided a method ofmonitoring the presence of one or more chromophores in a sample ofbiological tissue, which method comprises

-   illuminating an area of such tissue sample by projecting light from    a light source,-   receiving light remitted by the illuminated area of tissue at a    photo-receptor, spectroscopically analyzing the remitted light,-   and comparing variations in the intensity and spectral    characteristics of the remitted light with respect to the intensity    and spectral characteristics of the projected light and with data    representing a datum sample of intensity and spectral    characteristics, and emitting a control signal in response to any    such variations.

In some embodiments, the invention is applied for the endoscopicmonitoring of the presence of one or more said chromophores in thetissue sample, but the invention is particularly apt for a whollynon-invasive analysis of tissue structure.

The invention in particular includes a method of non-invasivelyanalyzing tissue structure, comprising the steps of:

-   (i) measuring red or infrared radiation from at least one location    in an area of tissue under investigation so as to give an indication    of any layered structure in said area;-   (ii) measuring the tissue color co-ordinates at said at least one    location in said area of tissue;-   (iii) using data obtained in measuring steps (i) and (ii) to    calculate corrected tissue color co-ordinates in respect of the or    at least one said area which corresponds to a predetermined    thickness of said layered structure, and;-   (iv) comparing the corrected tissue color co-ordinates obtained in    step (iii) with a reference color co-ordinate range for healthy    tissue having a layered structure of the same predetermined    thickness.

Such method may be used for locating and measuring the properties of anabnormality in a layered structure, for example a collagen-rich layer,such as skin. The light measured in step (i) above suitably extendsacross the UV and/or visible and/or IR regions.

Such method most preferably further comprises the additional step of;

-   (v) identifying corrected tissue color co-ordinates which lie    outside the reference color co-ordinate range.

Such method also preferably further comprises the additional steps of;

-   (vi) comparing the degree of deviation of the corrected tissue color    co-ordinates which lie outside the reference color co-ordinate range    with generalized levels of deviation from a reference color    co-ordinate range known to be associated with differing    abnormalities in said tissue, and;-   (vii) using the tissue color co-ordinates to assess the degree of    abnormality of said tissue.

In alternative preferred embodiments, the method comprises theadditional steps of

-   (vi) calibrating the corrected tissue color co-ordinates with the    corrected tissue co-ordinates of at least one tissue location having    color co-ordinates lying within said reference color co-ordinate    range for normal tissue;-   (vii) using the tissue color co-ordinates to assess the degree of    abnormality of said tissue.

Suitably, said calibration in step (vi) includes estimating the level ofepidermal melanin at said location by reference to epidermal melaninlevels calculated within at least one normal skin region adjacent saidlocation. Alternatively said calibration in step (vi) may includemeasuring epidermal melanin levels at said location by assessing thedeviation at the blue end of the spectrum at said location from thereference color co-ordinate range for normal skin.

The invention further includes a method of mapping the papillary surfaceof an area of the dermis which comprises illuminating the surface of theskin over that area with light and monitoring the intensity of the lightremitted from along at least one line or sequence of points, the lighthaving a wavelength sufficiently far into the infra-red that itsabsorption by melanin and blood is negligible, or having at least twowavelengths of which at least one is in excess of 600 nm and derivingtherefrom a theoretical intensity of remitted light which is independentof the presence of melanin or blood, and from the remitted lightintensity deriving a signal corresponding to the concentration ofcollagen within the papillary dermis along the or each line or at eachpoint, and producing a contoured image in which the apparent elevationof any point is dependent upon the strength of such signal.

The invention extends to apparatus which may be used for performing amethod as herein defined, and accordingly includes apparatus formonitoring the presence of one or more chromophores in a biologicaltissue sample, which apparatus comprises

-   a light source for projecting light to illuminate an area of such    tissue sample,-   a photo-receptor for receiving light remitted by the illuminated    area of tissue, and a spectroscopic analyzer for monitoring the    remitted light,-   a comparator for comparing variations in the intensity and spectral    characteristics of the remitted light with respect to the intensity    and spectral characteristics of the projected light at different    wavelengths and with data representing a datum sample of intensity    and spectral characteristics of light and a signal emitter for    emitting a control signal in response to any such variations.

The invention extends to apparatus for non-invasively analyzing skinstructure, comprising:

-   means for projecting UV and/or visible and/or red and/or infrared    radiation onto an area of skin under investigation,-   measuring means for measuring remitted red or infrared radiation    from at least one location over said area of skin so as to give an    indication of the collagen thickness in said area;-   skin color co-ordinate measuring means for measuring the skin color    co-ordinates at said at least one location in said area of skin;-   calculating means for using data obtained in measuring steps (i)    and (ii) to calculate corrected skin color co-ordinates in respect    of the or at least one said area which corresponds to a    predetermined amount of collagen, and;-   color comparison means for comparing the corrected skin color    co-ordinates obtained in step (iii) with a reference color    co-ordinate range for skin with the same collagen content.

The invention further extends to apparatus for mapping the papillarysurface of an area of the dermis which comprises a light sourceilluminating the surface of the skin over that area with light whicheither has a wavelength sufficiently far into the infra-red that itsabsorption by melanin and blood is negligible, or which has at least twowavelengths of which at least one is in excess of 600 nm, means formonitoring the intensity of the light remitted along at least one lineor sequence of points, and deriving therefrom an intensity ortheoretical intensity of remitted light which is independent of thepresence of melanin or blood, and means for deriving a signal from theremitted light intensity corresponding to the concentration of collagenwithin the papillary dermis along the or each line or at each point, andfor producing a contoured image in which the apparent elevation of anypoint is dependent upon the strength of such signal.

The present invention is based on the findings reported by Symon D'OCotton in “Do all human skin colors lie on a defined surface within LMSspace?”, University of Birmingham Technical Report, 30 Dec. 1995. Thedisclosure of such Technical Report is included herein by reference. Inthis Technical Report, the relation between healthy skin and the colorof the skin represented in LMS, a particular color space, is reported,and it discloses that, for healthy skin, the coloration, regardless ofrace or amount of tanning, lies on a defined curved surface within athree-dimensional color space. This, if used with a correct colormeasurement system, can measure and quantify the amount of melanin andblood and other chromophores at any particular point at which thismeasurement is made. If the skin is sampled as an image, thencorresponding images showing the variation of blood and melanin acrossthe skin can be obtained. In the above Technical Report, it is disclosedthat melanin can sometimes penetrate into the dermis producing thecharacteristic hues of melanoma and that this melanocytic descent hasbeen quantified by Clark et al (“The Histogenesis and BiologicalBehavior of Primary Human Malignant Melanomas of the Skin”, CancerResearch, 29, 1989) into five levels of tumor invasion, in which level 1corresponds to confinement within the epidermis, level 2 corresponds toinvasion into the papillary dermis, etc. In an alternative system, theextent of tumor invasion in mm from the cornified layer is expressed asthe Breslow thickness. The above Technical Report also acknowledgesthat, in the case of melanoma, CD Neville (“Melanoma: Issues ofImportance to the Clinician”, British Journal of Hospital Medicine,March 1985) discloses the existence of a strong relationship betweenthis level of invasion and prognosis. However, the above TechnicalReport does not disclose in detail any method suitable for taking thenecessary measurements.

The invention is presently believed to be of particular value in themonitoring of color variations in epithelial and sub-epithelial tissues,and it is accordingly preferred that said datum sample represents theintensity and spectral characteristics of light remitted by a sample ofepithelial or epithelial and sub-epithelial tissue. In particular, theinvention is useful for investigating skin tissue histology, and forthis purpose, it is desirable that said datum sample represents theintensity and spectral characteristics of light remitted by a sample ofskin. It is desirable that said datum sample represents the intensityand spectral characteristics of light remitted by a sample of tissue ofknown structure. Alternatively, or in addition, it is desirable thatsaid datum sample represents the intensity and spectral characteristicsof remitted light as calculated from a mathematical optical model of thetissue.

The apparatus and method of the present invention may be utilized formonitoring the presence of a wide variety of chromophores in the skinand in other biological tissue. It is possible to derive data relatingto the presence, depth, and concentration of a wide range ofchromophores, depending on measurements being made at particularwavelengths. These wavelengths may readily be selectable by lightfilters which may be substituted into the light path, or the analyzermay be constituted by a spectroscope. The filters may be broad bandfilters or narrow band filters as appropriate for the analysis to beundertaken.

Examples of particular chromophores whose presence may be monitoredinclude: melanin, blood, haemoglobin, oxy-haemoglobin, bilirubin, tattoopigments and dyestuffs, keratin, collagen and hair.

It is to be understood that using the method of the invention, it ispossible to reconstruct a full 3D model of the architecture of the skinor other tissue which conveys information grossly comparable to thatavailable through microscopical examination of biopsied tissue.

In particular, having regard to the examination of skin, it has beenfound that the papillary dermal skin thickness can change markedly withsome skin lesions which are not otherwise of concern. This throws thecoloration of the skin off the surface of predicted coloration and socan give rise to false measurements of the histology of such skinlesions. It is for this reason that papillary dermis thickness isusually measured first, and subsequent calculations are based on theskin color co-ordinates corrected to a predetermined papillary dermisthickness. Any arbitrary value for this thickness may be chosen, such as2.0×10⁻⁴ m which is the average value for healthy human skin.

The thickness of the papillary dermis may be obtained by utilizing amethod which will be described below with reference to FIG. 1.

In a preferred embodiment, the reference color co-ordinate range fornormal skin at the predetermined papillary dermis thickness is obtainedas disclosed in the above-mentioned Technical Report as a curved surfacelying within a three-dimensional color space, with one of the boundingaxes relating to the amount of melanin within the epidermis and theother relating to the amount of blood within the dermis. When an areacontaining dermal melanin is located, i.e. points do not lie on thenormal color surface, the epidermal melanin value within. this area isestimated by either reference to the reference color co-ordinate rangefor normal skin within regions identified as normal, or by reference tothe epidermal melanin levels calculated within normal regions adjacentto said area containing dermal melanin. This value is then used with thecorrected color co-ordinates of the abnormal region at the samepredetermined papillary dermis thickness to compute invasion depth andconcentration of dermal melanin. The corrected skin color co-ordinatesfor the area of skin under investigation may be calibrated to valuesequivalent to zero epidermal melanin. Instead of using LMS color space,it is possible to use any other color space, for example, the RGB colorspace or a UV G IR color space.

The dermis contrasts strongly in structure to that of the epidermis,being highly vascular, containing many sensory receptors and being madelargely from collagen fibers to provide the essential structure of theskin. Between the epidermis and the dermis, the junction presents anextremely uneven boundary with finger-like dermal protrusions calleddermal papillae projecting towards the skin surface. The dermis can besplit into two further histologically distinct layers, the papillarydermis and the reticular dermis within which the structure of thecollagen fibers differs significantly. The papillary dermis is situateddirectly below the epidermis and within which the collagen exists as afine network of fibers. This is in contrast with the reticular dermiswhere the collagen fibers are aggregated into thick bundles which arearranged nearly parallel to the skin surface. In the case of melanininvasion of the papillary dermis, there is a layer containing blood,melanin and collagen, a layer containing either blood and collagen ormelanin and collagen, depending upon whether melanin has passed theblood layer; and a layer containing just collagen. The differentthicknesses of these layers, the amount of blood and the concentrationof dermal melanin along with the amount of melanin in the overlyingepidermis affect the remitted light. This can be modeled by calculatingthe net effect of these three layers for the differing parametersoutlined.

A mathematical model describing the optics of the skin has beendescribed in the above mentioned Symon D'O Cotton's Technical Report,whose disclosure has been included herein by reference, and this modelcan be extended to predict coloration of skin containing dermal descentof melanin.

As can be seen from FIG. 2, there are now four distinct layers withinthe dermis which can combine to construct a simple model, 1) a layerwithin the upper papillary dermis containing no melanin, 2) a layerwithin the upper papillary dermis containing melanin, 3) a layer withinthe lower papillary dermis containing melanin, 4) a layer within thelower papillary dermis containing no melanin.

It should also be noted that the condition of melanin existing up to thedermo-epidermal junction is facilitated by allowing the thickness oflayer 1 to be zero and likewise melanin can exist up to thepapillary-reticular dermis boundary by setting the thickness of layer 4to be zero.

In computing a model to predict this coloration it is useful to makenote of the fact that, as discussed in section 2.1 of the TechnicalReport, the amount of back scatter due to melanin can be considerednegligible. Therefore, in the same manner that it was possible to applythe Kubelka-Munk theory to the papillary dermis (section 3.2.2 of theTechnical Report), to compute the coloration of sections of papillarydermis containing blood, where the back scattering component of bloodwas considered negligible, it is possible to compute the coloration ofsections containing melanin. In this situation ζ(λ) (scatteringcoefficient) remains dependent only on wavelength whilst a (fraction ofradiation absorbed per unit path length) becomes α(λ,ρ,φ) where φrepresents the density of dermal melanin within that layer. Further,following the proof given in equation (17) of the Technical Report,α(λ,ρ,φ) can be shown to be the sum of α_(iv)(λ), α_(b)(λ) and α_(m)(λ),where α_(m)(λ) is the absorption coefficient of melanin. From the aboveit is possible to calculate R and T (diffuse radiation and transmissionrespectively). For simplicity of notation it is helpful to consider R₁and T₁ where,R ₁(λ,ρ,φ,d _(n))=R(β(k(α(λ,ρ,φ)),s(ζ(λ))), K(k(α(λ,ρ,φ)), s(ζ(λ))),d_(n))  Eqn.1andT ₁(λ,ρ,φ,d _(n))=T(β(k(α(λ,ρ,φ))s(ζ(λ))),K(k(α(λ,ρ,φ)), s(ζ(λ))),d_(n))  Eqn.2where d_(n) is the layer thickness.

As was shown in section 3.2.3 of the Technical Report, two-layer systemscan be combined to produce the total remitted and transmitted light forthe dermis resulting in equation (20) of the Technical Report.

This can be simplified using the geometric series $\begin{matrix}{{a + {ar} + {ar}^{2} + {ar}^{3} + \ldots} = {{{\frac{a}{1 - r}\quad{if}} - 1} < r < 1}} & {{Equation}\quad 3}\end{matrix}$to $\begin{matrix}{{R_{1{total}}\left( {\lambda,\rho_{ud},\rho_{ld},d_{ud},d_{ld}} \right)} = {{R_{1{ud}}\left( {\lambda,\rho_{ud},d_{ud}} \right)} + \frac{\begin{matrix}{T_{1{ud}}\left( {\lambda,\rho_{ud},d_{ud}} \right)}^{2} \\{R_{1{ld}}\left( {\lambda,\rho_{ld},d_{ld}} \right)}\end{matrix}}{\begin{matrix}{1 - {R_{1{ud}}\left( {\lambda,\rho_{ud},d_{ud}} \right)}} \\{R_{1{ld}}\left( {\lambda,\rho_{ld},d_{ld}} \right)}\end{matrix}}}} & {{Eqn}\quad 4}\end{matrix}$Similarly, T_(1total) can be shown to be $\begin{matrix}{{T_{1{total}}\left( {\lambda,\rho_{ud},\rho_{ld},d_{ud},d_{ld}} \right)} = \frac{\begin{matrix}{{T_{1{ud}}\left( {\lambda,\rho_{ud},d_{ud}} \right)} \times} \\{T_{1{ld}}\left( {\lambda,\rho_{ld},d_{ld}} \right)}\end{matrix}}{\begin{matrix}{1 - {R_{1{ud}}\left( {\lambda,\rho_{ud},d_{ud}} \right)}} \\{R_{1{ld}}\left( {\lambda,\rho_{ld},d_{ld}} \right)}\end{matrix}}} & {{Equation}\quad 5}\end{matrix}$These equations can be extended, as is shown by W an et al. [1981], toan n layered system resulting in values for R_(l2 . . . n) andT_(l2 . . . n), of $\begin{matrix}{R_{\overset{.}{12}\quad\ldots\quad n} = {R_{12\quad\ldots\quad{({n - 1})}} + \frac{T_{12\quad\ldots\quad{({n - 1})}}^{2}R_{n}}{1 - {R_{12\quad\ldots\quad{({n - 1})}}R_{n}}}}} & {{Equation}\quad 6}\end{matrix}$ $\begin{matrix}{T_{\overset{.}{12}\quad\ldots\quad n} = \frac{T_{12\quad\ldots\quad{({n - 1})}}T_{n}}{1 - {R_{12\quad\ldots\quad{({n - 1})}}R_{n}}}} & {{Equation}\quad 7}\end{matrix}$

This system of equations can therefore compute the total remitted andtransmitted light from an n layered system of arbitrary complexityprovided that the thickness and composition of the layers is specified.

For the four-layer system shown in FIG. 2, this results in a value forthe total light remitted and transmitted from the dermis dependent on λ,ρ_(ud), ρ_(ld, d) _(ud), d_(ld), d_(l2), φ_(l2), d_(l3) and d_(l3) whered_(l2) and d_(l3) are the thickness of layers 2 and 3 whilst φ_(l2) andφ_(l3) are their corresponding melanin densities. The thickness of layer1 and layer 2 do not need to be explicitly defined as they are simplyd_(ud)-d_(l2) and d_(ld)-d_(l3) respectively; similarly φ_(l1) andφ_(l4) are zero by definition. A further simplification is possible ifit is assumed that φ_(l2)=φ_(l3) leading to a single value of φ for thedermis.

The results of these equations can be combined with the predicted lighttransmitted by the epidermis in the same manner as that discussed insection 3.3 of the Technical Report, thus leading to the followingdescription of total remitted, S_(rd), and transmitted S_(td).S _(rd)(λ,ρ_(ud),ρ_(ld) ,d _(ud) ,d _(ld) ,d _(l2) ,d _(l3) ,φ,d _(m))=R _(2total)(λ,ρ_(ud),ρ_(ld) ,d _(ud) ,d _(ld) ,d _(l2) ,d _(l3),φ)θ(λ,d _(m))² S(λ)  Eqn. 8S _(td)(λ,ρ_(ud),ρ_(ld) ,d _(ud) ,d _(ld) ,d _(l2) ,d _(l3) ,φ,d _(m))=T_(2total)(λ,ρ_(ud),ρ_(ld) ,d _(ud) ,d _(ld) ,d _(l2) ,d _(l3),φ)θ(λ,d_(m)) ² S(λ).  Eqn. 9

These can be used to predict the value of the corresponding LMSprimaries $\begin{matrix}{{L\left( {\rho_{ud},\rho_{ld},d_{ud},d_{ld},d_{12},d_{13},\phi,d_{m}} \right)} = {\int_{0}^{\infty}{{R_{2{total}}\left( {\rho_{ud},\rho_{ld},d_{ud},d_{ld},d_{12},d_{13},\phi} \right)}{\theta\left( {\lambda,d_{m}} \right)}^{2}{S(\lambda)}{S_{L}(\lambda)}\quad{\mathbb{d}\lambda}}}} & {{Eqn}\quad 10}\end{matrix}$ $\begin{matrix}{{M\left( {\rho_{ud},\rho_{ld},d_{ud},d_{ld},d_{12},d_{13},\phi,d_{m}} \right)} = {\int_{0}^{\infty}{{R_{2{total}}\left( {\rho_{ud},\rho_{ld},d_{ud},d_{ld},d_{12},d_{13},\phi} \right)}{\theta\left( {\lambda,d_{m}} \right)}^{2}{S(\lambda)}{S_{M}(\lambda)}\quad{\mathbb{d}\lambda}}}} & {{Eqn}\quad 11} \\{{S\left( {\rho_{ud},\rho_{ld},d_{ud},d_{ld},d_{12},d_{13},\phi,d_{m}} \right)} = {\int_{0}^{\infty}{{R_{2{total}}\left( {\rho_{ud},\rho_{ld},d_{udd},d_{ld},d_{12},d_{13},\phi} \right)}{\theta\left( {\lambda,d_{m}} \right)}^{2}{S(\lambda)}{S_{S}(\lambda)}\quad{\mathbb{d}\lambda}}}} & {{Equation}\quad 12}\end{matrix}$

A further generalization can be made to any primary, P_(n), leading tothe following equation where S_(n) defines the spectral response of thatprimary. $\begin{matrix}{{P_{n}\left( {\rho_{ud},\rho_{ld},d_{ud},d_{ld},d_{12},d_{13},\phi,d_{m}} \right)} = {\int_{0}^{\infty}{{R_{2{total}}\left( {\rho_{ud},\rho_{ld},d_{ud},d_{ld},d_{12},d_{13},\phi} \right)}{\theta\left( {\lambda,d_{m}} \right)}^{2}{S(\lambda)}{S_{P_{n}}(\lambda)}\quad{\mathbb{d}\lambda}}}} & {{Equation}\quad 13}\end{matrix}$

This equation can then be used to generate the expected coloration ofhuman skin exhibiting dermal descent of melanin.

The result of this analysis is that it is possible for the samecoloration to result from different combinations of the aboveparameters. This complicates the measurement of the dermal invasion ofmelanin, (but not identifying the presence of any dermal melanin).Indeed, to obtain this measurement, it is necessary to know the amountof melanin in the overlying epidermis. However, at points where dermalinvasion has taken place, this parameter is difficult to determinesimply by comparing color co-ordinates of the abnormal location withcolor co-ordinates for healthy skin. It is for this reason that, in thepresent invention, regions where dermal melanin exists are identified byreference to a reference color co-ordinate range for healthy skin, andthen the color co-ordinates of these regions are compared with the colorco-ordinates at one or more normal skin locations. If said normal skinlocations are adjacent to the region where dermal melanin exists, it issufficient to use the epidermal melanin levels calculated for suchnormal skin locations to estimate the epidermal melanin levels at theregion where dermal melanin exists. Alternatively, it is possible toperform a measurement of the epidermal melanin levels within areas ofthe skin where the presence of dermal melanin has been identified, byassessing the deviation in coloration at the blue end of the spectrum,from the reference color co-ordinate range for normal skin due to thepresence of such dermal melanin. At the blue end of the spectrum, theincrease in such deviation quickly slows with increasing depth ofmelanin penetration until a “saturation point” is reached. By assumingthat the depth of melanin penetration within the dermis is large enoughfor such saturation to have occurred, an estimate of the deviation fromthe reference color co-ordinate range for normal skin can be made. Thisestimate allows a calculation to be made of the skin coloration assumingno dermal melanin, and therefore by reference to the color co-ordinaterange for normal skin, of the level of epidermal melanin. It is withinthe scope of the present invention to measure the epidermal melaninlevels directly, for example using polarized light, and to incorporatesuch measurements in the measuring step (ii) above.

By any of the above methods, the effect of what would have been thenormal epidermal melanin level in the abnormal skin location can betaken into account, thereby enabling a more accurate determination ofmelanin descent.

By comparing the values of the skin image represented in a certain colorspace with theoretically calculated values covering all possible amountsof blood, dermal melanin penetration and melanin concentration withinthe same color space, the values of those three parameters can beobtained for every point in the image. Since the papillary dermisthickness and epidermal melanin content are known, it is possible tocompute a detailed three-dimensional reconstruction of the top layers ofhuman skin. This is of great potential interest to the medicalprofession and enables routine examination of the internal structure ofliving skin just as X-rays, NMR and ultrasound are used for examiningother parts of the body. Other living tissue accessible by endoscope mayalso be examined, as may tissue which has been removed from a living ordead body. It is also within the scope of the invention to acquire theinfra-red and/or visible images using lasers of different wavelengths orby using spectral analysis.

Deviations from the normal color surface may be due to unusualchromophores or constituents in the papillary dermis or epidermis: thenature and size of deviation gives information on the depth andconcentration of these chromophores. Within pigmented skin lesions aparticularly interesting chromophore is melanin, and its depth,concentration and distribution give useful information for the diagnosisof many skin conditions.

To assess such deviations it is important to understand that thedeviation from the normal color surface is relative to the point on thatsurface relating to the amount of epidermal melanin. As such, knowledgeof the amount of epidermal melanin allows examination in detail of theexact deviation which is due only to the chromophore in question. Forinstance, dermal melanin can vary in both its depth and concentrationwith a similar coloration or spectral properties being obtained fromvarying amounts of epidermal melanin and combinations of depth andconcentration. This complicates the recovery of information relating tothe depth and concentration of dermal melanin. If, however, the amountof epidermal melanin were known, an allowance could be made for it, or ameasurement of the deviation could be obtained from the relevantposition on the normal color surface where skin with such an amount ofepidermal melanin would lie.

Such a measurement of epidermal melanin within regions of dermal melaninis complicated by the effect of the dermal melanin on the spectralremittance of the skin. However, a number of methods can be used toaccount for it:

1) Ascertain the amount of epidermal melanin in a region of skinadjacent the region with dermal melanin and interpolate this quantityinto the melanistic region. This may be performed at a single locationor at a number of points, e.g. surrounding the area of dermal melanin.Although such a measure is an interpolation it can give a good estimateallowing processing to proceed as described above.

2) If a technique is used as described with reference to Equation 32below, a detector which is blind to epidermal melanin can be used toinvestigate such regions. This utilizes a technique where epidermalmelanin is assessed using a set of detectors and algorithms which have azero or constant response to varying amounts of epidermal melanin.Variations in dermal melanin, or other chromophores, however will stillelicit a response allowing the construction of look-up tables relatingthe output of such sensors to variations in these constituents. Such atechnique would not suffer from the effects of overlying epidermalmelanin as it is blind to it.

3) To obtain highly accurate measures of dermal melanin penetration andconcentration, it is preferable to ascertain the amount of epidermalmelanin, however, it is possible to obtain useful information withoutsuch a correction. By varying the amount of dermal melanin andconcentration either mathematically or by biological studies it ispossible to measure the maximum possible deviation from the surface ofnormal skin coloration. In general as the ‘corrected for collagenvariations’ skin coloration or spectral properties nears this maximumthe deeper the dermal melanin lies in the papillary dermis. Therefore byascertaining the ratio of distance from the surface of normal skincoloration to such a measure relative to its distance from the maximalvalues a measure of depth can be made.

It is possible to use a computer programmed with the above algorithms toperform the actual calculations. However, before these calculations canbe performed, an image of the area of skin under investigation must berepresented in the same color space as for the normal skin referencecolor co-ordinate range. This can be done in a number of ways. In oneway, the skin color co-ordinates are acquired from an image using thesame lighting conditions and a CCD camera calibrated in the same way asthat used to produce the healthy skin reference color co-ordinate range.Alternatively, if exactly the same lighting conditions are not used, awhite standard or other appropriate correction factor can be used toallow calibration of the image within the software. As a furtheralternative, a color image can be acquired using a color photographicfilm which is then digitized. This can be performed using either exactlythe same lighting conditions and a calibrated set-up or again with theinclusion of a white standard or other appropriate correction factor. Itis within the scope of this invention to obtain both the infra-red andvisible images with a single digital camera or to calculate the value ofthe necessary primaries through the use of spectroscopy.

A method and apparatus according to the invention are valuable inproviding information to a clinician on which the clinician may base adiagnosis or a course of treatment and the apparatus may be used forcontrolling the treatment and in some cases for giving an indication ofwhether the treatment may be effective or not. Some preferredembodiments of the invention are applied for controlling a treatmentwhich involves the irradiation of a region of tissue with treatmentlight of predetermined spectral characteristics, wherein the absorptioncharacteristics of tissue supervening the region to be treated for thetreatment light are measured and used in calculating a required exposureof the tissue to the treatment light, or for predicting the outcome of atreatment which involves the irradiation of a region of tissue withtreatment light of predetermined spectral characteristics, wherein theabsorption characteristics for the treatment light of the tissue regionto be treated and of tissue supervening the region to be treated aremeasured and used in calculating a required therapeutically effectiveexposure of the tissue to the treatment light, and the required exposureand the absorption characteristics of the supervening tissue are used topredict potential destruction or scarring of the supervening tissue bysuch exposure.

For example, haemangioma (so-called port-wine stains, due to an abnormaldistribution of blood vessels) may be diagnosed by straight-forwardvisual inspection, and it is well known to treat the condition by laserto cauterize the blood vessels. Typically treatment for such a problembegins with the firing of a series of “test shots” by a laser atdifferent powers to establish the minimum power necessary to cauterizethe blood vessels. That power will depend on the depth and the size ofthose vessels, and these may vary over the extent of the lesion. Thetest powers are chosen by the clinician having regard to his skill andpast experience. This technique suffers from a number of disadvantages.It is not very reliable since the depth and the size of the bloodvessels may vary over the extent of the lesion. It is time consumingsince the results of the test need to be assessed after a healing time.And the patient is left in a state of uncertainty during that time. Thisuncertainty is exacerbated due to the fact that a too intense laserirradiation will result in burning of the skin and consequent permanentscarring. In the cases of up to about one third of patients, theintensity of irradiation which would be required to cauterize theoffending blood vessels is actually so high that there would be aserious risk of scarring and the treatment is accordinglycontra-indicated.

The present invention can be used to establish not only the amount ofblood present, and thus give an indication of the amount of bloodvessels required to be cauterized, but also the depth of those vesselsbeneath the surface of the skin. In addition the invention can becarried out in such a way as to calculate the amount of melanin and anyother unwanted energy-absorbing chromophores, such that through aknowledge of the spectral absorption characteristics of thesechromophores and the spectral characteristics of the incident treatmentlight an estimate can be made of the energy absorption of thesechromophores and therefore the amount of unwanted heating of thesupervening or other tissue that would be unavoidably irradiated duringtreatment. The intensity of laser irradiation needed to cauterize agiven amount of blood vessels is known from past experience or can beestablished, and the absorption characteristics of human skin inrelation to the laser radiation of a given wavelength can readily beestablished (indeed handbooks supplied with medical lasers tend tocontain this information).

Thus by making use of the present invention, light remitted from thestain can be analyzed to give an indication of the melanin content ofthe epidermis (which governs its absorption coefficient) and of thedepth and concentration of the offending blood vessels, and a predictioncan be made there and then as to the intensity of laser irradiationwhich will be required to effect a satisfactory treatment and whetherthat intensity would give an acceptably low risk of permanent scarring.Further this assessment may be made at as many points over the extent ofthe stain as are thought necessary. Not only that, but the output signalfrom the apparatus may be used to control firing of a laser. Thus thepower output of the laser may be varied as it is directed over theextent of the lesion. Thus the laser may be controlled to give theminimum effective power dissipation over the various increments of thelesion. Parts only of the area of the lesion could be treated if thatwould give a cosmetically acceptable result. And if the lesion was sosevere that it was unsuitable for laser treatment, the patient could betold immediately and would not face some weeks of uncertainty.

The present invention can thus also be used to predict the outcome of agiven course of treatment. This may be achieved by predicting the effectof treatment on the quantity of the chromophore to be eliminated orstimulated. This can be done by correlating experimentally the energyreaching the chromophore with the effect produced by that energy, and bymeasuring the structure and chromophore before and following treatment.Using this predictive data, the predicted change in a tissue sample canbe calculated by measuring that sample and calculating the energyreaching that chromophore, relating that energy to a predicted change inthat chromophore through the experimental correlation, and thenrecalculating the effect of that revised chromophore level on the tissueappearance using the optical model described in this document.

Similar considerations apply in the case of removing tattoos by thedestruction of the pigments used to make them, and in the destruction ofmelanin/melanocytes during the removal of moles. Similar considerationsalso apply in the case of the stimulation of collagen growth using lightenergy to stimulate collagen-producing cells.

The removal of hair by laser cauterization of the hair bulb may also becontrolled by apparatus according to the invention. Hair consists ofkeratin and its color (and thus light energy absorption characteristics)is due to the presence of melanin. The hair bulb is located in or belowthe reticular dermis. Using the present invention it is possible todetermine the absorption characteristics of the skin layers which wouldhave to be penetrated by laser radiation aimed to destroy the hair bulb.The absorption characteristics of the hair bulb can be measured orcalculated from a measurement of the melanin content of the hair, andthe amount of energy which would have to be absorbed by the hair bulb todestroy it can also be determined, in vitro if necessary. From thisinformation, it is possible to calculate the energy which would requireto be dissipated by the laser, and it would accordingly be possibleeither to give a minimum energy dosage, or to predict that the minimumrequired dosage was so high that permanent scarring would result andthat the treatment should accordingly not be carried out.

It will be appreciated that the output signal generated by the use ofthe invention will represent an average value over the extent of thearea monitored: this will plainly be no greater than the size of thelight spot which is illuminated, and its size may also be determined bythe size of the photoreceptor. Means may be provided for varying themonitored area if desired, for example from a spot 0.1 mm or less (e.g.0.01 mm) to 10 cm or more in diameter. This can be extended to providean image of an area by providing the analysis at a number of locations.This can easily be achieved by the use of a digital camera.

To achieve these results, the system measures the light remitted fromskin and compares it with the incident light at a number of wavelengthsor wavelength bands. These measurements can be performed using anyconvenient means including filters or a spectrometer and they allowquantification of the quantities and position, including distancerelative to the dermo-epidermal junction, of chromophores such ascollagen, melanin, blood and keratin. Indeed these measurements can beperformed on any substance assuming its absorbency and reflectivity oflight are known. “Spectral measurement” is used to denote measurement ofthe light remitted from human skin whether by the use of a spectrometeror sub-sampling through filters which can be placed in the path of theincident or remitted light. The spectral measurements can be performedat one or more points. A collection of points whether gatheredsimultaneously or not can also be combined to form an image showing themeasurements over the skin.

The spectral remittance of light from human skin can be calculated givenknowledge of the quantity and position of substances within it. Suchcalculations can be performed using a variety of mathematical meansincluding Monte Carlo modeling and the Kubelka-Munk theory, generating avalue for P_(n) where $\begin{matrix}{\begin{matrix}{P_{n}\left( {\rho_{1},\rho_{2},{\rho_{3}\quad\ldots\quad\rho_{n}},d_{1},d_{2},{d_{3}\quad\ldots\quad d_{n}},}\quad \right.} \\\left. {\phi_{m1},\phi_{m2},{\phi_{m3}\quad\ldots\quad\phi_{mn}},d_{m},v,\kappa} \right)\end{matrix} = \frac{\begin{matrix}{\int_{0}^{\infty}{R\left( {\rho_{1},\rho_{2},{\rho_{3}\quad\ldots\quad\rho_{n}},d_{1},d_{2},{d_{3}\quad\ldots\quad d_{n}},} \right.}} \\{\left. {\phi_{m1},\phi_{m2},{\phi_{m3}\quad\ldots\quad\phi_{mn}},v} \right){\theta\left( {\lambda,d_{m},\kappa} \right)}^{2}{S(\lambda)}{S_{P_{n}}(\lambda)}{\mathbb{d}\lambda}}\end{matrix}}{\int_{0}^{\infty}{{S(\lambda)}{S_{P_{n}}(\lambda)}{\mathbb{d}\lambda}}}} & {{Equation}\quad 14}\end{matrix}$in which:

P_(n) represents the calculated or measured ratio of remitted toincident light for a particular wavelength function or filter S_(P) _(n)(λ) and incident light S(λ). θ represents the light absorbed within theepidermis with dm representing the quantity of epidermal melanin and κthe amount of keratin. R represents the ratio of light remitted from thedermis to light incident on the dermis, with ρ₁,ρ₂,ρ₃ . . . ρ_(n)representing the quantity of blood within n layers within the dermis,parallel with the skin surface and of thicknesses d₁,d₂,d₃ . . . d_(n).Within these layers, φ_(m1),φ_(m2),φ_(m3) . . . φ_(mn) represent thequantity of melanin within the dermis and v the thickness of thepapillary dermis. P_(n) can also be obtained through measurements onreal skin rather than by calculation.

As discussed the position within the dermis and concentration of bloodis of importance to the calibration and use of medical lasers. Theposition of such blood will effect the remitted light from the skingenerally causing the skin color to become more purple as the depth ofblood vessels increases.

To ascertain non invasive information regarding blood position andconcentration the spectral composition of light remitted from skin canbe ascertained as above for a representative sample of possible bloodquantities and blood depths. It is also necessary to generate thepossible set of remitted light measurements relating to variations inother parameters such as epidermal melanin, dermal melanin, papillarydermal thickness and keratin. As such an N dimensional search space isgenerated where N corresponds to the number of different constituentsand blood and melanin planes considered. This analysis can be extendedto include any other constituents such as tattoo pigment. For analysisof skin this may have to include spectral measurements within theinfrared portion of the electromagnetic spectrum as well as the visible.

Measurements of the spectral remittance from skin to be examined arethen compared with the data within the N dimensional search space withthe closest match indicating the constituents of the skin. The data forthese comparisons can either be performed as required or incorporatedinto pre calculated lookup tables.

Such an analysis may require a large search and it is possible forcertain combinations of constituents to generate the same spectralremittance and thus multiple solutions.

Another approach is to identify those constituents of skin about whichinformation can reliably be ascertained, quantify these and perform atransformation to the measured spectral remittance or data to which thisis to be compared.

This can for instance be achieved by first adjusting for variations inthe thickness of the papillary dermis in the manner described above. Asecond quantity that must also be assessed is the quantity of melaninwithin the epidermis. The accuracy to which this can be assessed has alarge influence on the accuracy to which the depth of blood within thedermis can be ascertained. However the presence of blood at differentdepths within the dermis markedly changes the remittance of light fromthe skin and so complicates the assessment of epidermal melanin levelsby standard spectroscopic means.

A solution to this problem assumes that the quantity of epidermalmelanin does not change markedly over the skin surrounding the lesionthus allowing interpolation from the surrounding areas. Such a techniquemay operate in certain lesions but the reliance that can be placed onthe results will be lowered. A second solution is to assess the levelsof epidermal melanin by a spectroscopic/light analysis method acceptingany inaccuracies due to the complicating factor of blood at differentdepths. Following either of these techniques the N dimensional space canbe reduced requiring only solutions to P_(nr) to be found where$\begin{matrix}{{P_{nr}\left( {\rho_{1},\rho_{2},\rho_{3},d_{1},d_{2},d_{3}} \right)} = {\int_{0}^{\infty}{{R_{nr}\left( {\rho_{1},\rho_{2},\rho_{3},d_{1},d_{2},d_{3}} \right)}{S(\lambda)}{S_{P_{n}}(\lambda)}\quad{\mathbb{d}\lambda}}}} & {{Equation}\quad 15}\end{matrix}$

As discussed inaccuracies in this measurement will adversely effect theassessment of blood position within the dermis thus lowering itsaccuracy.

A third solution is to use a detector which is “blind” to the effect ofmelanin within the epidermis. Such a detector would register zero, or aconstant value, when presented with melanin within the epidermis withdifferences in its value corresponding purely to the quantity andposition of other skin constituents. Such a detector would not requiretransformations to data based on measures for the amount of epidermalmelanin thus increasing accuracy. It is also possible to use such adetector in the generation of the N dimensional search space discussedpreviously.

The epidermal-melanin-blind detector renders the pigment melanineffectively transparent when it lies within the epidermis of the skin.Such a detector allows viewing of structures within the skin with theobscuring effect of epidermal melanin removed. The approach outlinedutilizes knowledge of the variation of light absorption by melaninwithin the epidermis with wavelength. The use of polarized light affordsadvantages in achieving melanin-blind results.

At a particular wavelength λ, let the ratio of remitted to incidentlight from skin be P(λ). If two wavelengths λ₁ and λ₂ are consideredthis leads to two values of P, P(λ₁) and P(λ₂).

Let R_(d)(λ,υ) represent the ratio of remitted to incident light frombloodless, melanin-free, normal dermis with a known quantity of collagenwithin the papillary dermis υ. Further let θ(λ,d_(m)) represent theratio of incident to transmitted light for melanin where d_(m)represents the quantity of melanin. It can be shown thatP(λ)=θ(λ,d_(m))²R_(d)(λ,υ), and thereforeP(λ₁)=θ(λ₁ ,d _(m))² R _(d)(λ₁,υ)  Equation 16andP(λ₂)=θ(λ₂ ,d _(m))² R _(d)(λ₂,υ)  .Equation 17

As further shown in “The optics of human skin” The Journal ofInvestigative Dermatology, (R. Anderson, B. Parrish & J. Parrish),θ(λ,d_(m)) can represented in the formθ(λ,d _(m))=e ^(−d) ^(m) ^(m(λ))  Equation 18where m(λ) is the spectral absorption coefficient of melanin. As suchEquations 16 and 17 becomeP(λ₁)=e ^(−2d) ^(m) ^(m(λ) ¹ ⁾ R _(d)(λ₁,υ)  Equation 19andP(λ₂)=e ^(−2d) ^(m) ^(m(λ) ² ⁾ R _(d)(λ₂,υ)  Equation 20

By taking the natural logarithm of both sides of the equations,equations 19 and 20 can be shown to equate toln P(λ₁)=ln e ^(−2d) ^(m) ^(m(λ) ¹ ⁾+ln R _(d)(λ₁,υ)  Equation 21andln P(λ₂)=ln e ^(−2d) ^(m) ^(m(λ) ² ⁾+ln R _(d)(λ₂,υ)   Equation 22which can be simplified to−2d _(m) m(λ₁)=ln P(λ₁)−ln R _(d)(λ₁,υ)=V ₁  Equation 23and−2d _(m) m(λ₂)=ln P(λ₂)−ln R _(d)(λ₂,υ)=V ₂  Equation 24

The proposition for an epidermal blind detector is that V₁−CV₂=0 where Cis a constant. For this to be true:−2d _(m) m(λ₁)+2Cd _(m)m(λ₂)=0  Equation 25and therefore $\begin{matrix}{C = \frac{m\left( \lambda_{1} \right)}{m\left( \lambda_{2} \right)}} & {{Equation}\quad 26}\end{matrix}$leading toln P(λ₁)−ln R _(d)(λ₁,υ)−C(ln P(λ₂)−ln R _(d)(λ₂, υ))=0  Equation 27

This discussion assumes bloodless skin where the only melanin presentexists in the epidermis. For real skin however this will often not bethe case, with blood, melanin in the dermis and keratin etc. beingpresent. In this situation an extra term E(λ) is introduced to the righthand side of Equations 21 and 22 representing the extra absorption, orindeed reflectance, introduced through the additional constituentsleading toln P(λ₁)=ln e ^(−2d) ^(m) ^(m(λ) ¹ ⁾+ln R _(d)(λ₁,υ)+ln E(λ₁)  Equation28andln P(λ₂)=ln e ^(−2d) ^(m) ^(m(λ) ² ⁾+ln R _(d)(λ₂,υ)+ln E(λ₂)  Equation29and thereforeln P(λ₁)−ln R _(d)(λ₁,υ)−C(ln P(λ₂)−lnR _(d)(λ₂,υ))=ln E(λ₁)−C lnE(λ₂)=F  Eqn 30

As P(λ₁) and P(λ₂) can be measured, C is known, and R_(d)(λ₁,υ) andR_(d)(λ₂,υ) can be calculated as disclosed in the International PatentApplication published as WO 98/22023, F can thus be calculated. Thevalue of F therefore indicates information about the extra terms E(λ₁)and E(λ₂) withF=C ln E(λ₂)−ln E(λ₁)  Equation 31and therefore $\begin{matrix}{{\mathbb{e}}^{F} = \frac{{E\left( \lambda_{2} \right)}^{C}}{E\left( \lambda_{1} \right)}} & {{Equation}\quad 32}\end{matrix}$

In summary, to operate the epidermal melanin blind detector measurementsP₁ and P₂, where P₁=P(λ₁)and P₂=P(λ₂), of skin are made and R₁ and R₂,where R₁=R_(d)(λ₁,υ) and R₂=R_(d) (λ₂,υ), are calculated. F is thencalculated from ln P₁−ln R₁−C(ln P₂−ln R₂) with its value givinginformation about pigments and components other than epidermal melanin.

The above analysis is based on the use of two measurements at twoseparate frequencies. However this can be extended to broad band filterswith values of m, the spectral absorption coefficient of melanin,calculated for each broad band filter.

As E(λ) relates purely to the change in remitted light, whether absorbedor reflected, without reference to the quantity of epidermal melanin orpapillary dermal thickness it is simple to calculate it for blood atdifferent quantities and depths within the dermis. The measured valuesof E(λ) can then be compared with these thus returning informationregarding the depth of blood vessels.

This approach can be extended to analyze constituents other than bloodwith the removal of epidermal melanin such as the examination ofkeratin, tattoo pigments, dermal melanin etc. Indeed the concept of amelanin blind detector can be extended to a blood blind detector, tattoopigment blind detector and indeed any constituent for which the lightreflectance and absorbency are known.

By allowing an accurate measurement of the depth and concentration ofblood vessels and other constituents, these measurements can then beused within Equation 14 thus allowing an accurate measurement ofepidermal melanin.

The knowledge gained regarding the position and constituents of humanskin can be utilized in Equation 14 to form a number of importantmeasures. For instance the percentage of light at any particularwavelength, or wavelength band, which is absorbed by epidermal melanincan be ascertained. This information can then be used to calculate thelikelihood of scarring occurring and thus allow the setting of a safemaximum intensity of light, whether through a laser or otherillumination device, that can be applied to the skin.

Further, the intensity, or percentage of light, passing through theentire papillary dermis can be ascertained. This is calculable using anequation similar to Equation 14 to result in the ratio, T, of incidentlight to light passing through the entire papillary dermis beingcalculated for a particular wavelength function or filter SPn(λ) andincident light S(λ). $\begin{matrix}{\begin{matrix}{T\left( {\rho_{1},\rho_{2},{\rho_{3}\quad\ldots\quad\rho_{n}},d_{1},d_{2},{d_{3}\quad\ldots\quad d_{n}},}\quad \right.} \\\left. {\phi_{m1},\phi_{m2},{\phi_{m3}\quad\ldots\quad\phi_{mn}},d_{m},v,\kappa} \right)\end{matrix} = \frac{\begin{matrix}{\int_{0}^{\infty}{T_{d}\left( {\rho_{1},\rho_{2},{\rho_{3}\quad\ldots\quad\rho_{n}},d_{1},d_{2},{d_{3}\quad\ldots\quad d_{n}},} \right.}} \\{\left. {\phi_{m1},\phi_{m2},{\phi_{m3}\quad\ldots\quad\phi_{mn}},v} \right){\theta\left( {\lambda,d_{m},\kappa} \right)}^{2}{S(\lambda)}{S_{P_{n}}(\lambda)}{\mathbb{d}\lambda}}\end{matrix}}{\int_{0}^{\infty}{{S(\lambda)}{S_{P_{n}}(\lambda)}{\mathbb{d}\lambda}}}} & {{Equation}\quad 33}\end{matrix}$

T_(d) represents the light transmitted through the papillary dermis andcan be calculated using a variety of mathematical means including MonteCarlo modeling and the Kubelka-Munk theory.

Such a measure is useful in quantifying the intensity that might impingeon a hair bulb and thus can be used to judge the efficacy of hairremoval by laser or other light source.

Similarly the intensity or percentage of light that reaches blood at aparticular depth can be ascertained and from this the quantity absorbedby the blood. Such a measure allows an assessment or calculation of theeffectiveness of the light in treating the blood vessels.

Following the quantification of the intensity of light impinging onvarious structures it is possible to ascertain, or quantify, the effectsuch an intensity will have on these structures. This may be performedthrough calculation or through analysis of previous treatments orthrough laboratory experiments. This knowledge then allows calculation,through Equation 14, of the expected appearance of the skin at either aparticular wavelength or wavelength band following the application ofsuch light. This information could, for instance, be used to generatecolor, RGB, representations of the expected result of a treatment whichwould be of great use in the planning of such treatment.

In preferred embodiments of the invention, the spectral analysis isundertaken at more than one, for example at least four, distinctwavelengths or wavelength bands, and in some preferred embodiments, suchanalysis is undertaken over the whole spectrum. In a simple constructionof apparatus, a filter wheel is placed between the source ofillumination, and the area of skin under inspection is successivelyilluminated using light of the desired different wavelengths orwavelength bands. In that case, all that is necessary is to measure theintensity of remitted light for each wavelength (band). Alternatively,white light may be used and the remitted light measured by aspectrometer to give values at each of a plurality of narrow wavelengthbands covering substantially the entire spectrum.

The use of narrow wavelength bands, whether due to filtering incident orremitted light or by use of a spectrometer, has advantages in certaincircumstances. For example, it may be desired to distinguish betweenarterial blood and venous blood. Arterial and venous blood have slightlydifferent spectral characteristics due to the presence or absence ofoxy-haemoglobin. Both oxy-haemoglobin and haemoglobin remit lightstrongly in the red, and their spectral curves in fact largely overlap.However, venous blood, without oxy-haemoglobin has a spectral curve witha domed peak, whereas arterial blood, due to the presence ofoxy-haemoglobin has a spectral curve with twin peaks separated by avalley. The use of two narrow band filters, one at a wavelengthcorresponding to one or other of those peaks, and one at a wavelengthcorresponding to the valley in the oxy-haemoglobin spectrum and acomparison of the intensity of light remitted at those wavelengths canthus determine the presence or absence of oxy-haemoglobin and thusdistinguish between venous and arterial blood.

The analysis of at least four different wavebands offers considerableadvantages over previous proposals, and allows the system to be used formeasuring a variety of different parameters which could not previouslyhave been unambiguously derived from the information given. For example,it allows the offset of chromophores to be measured. By offset, we meanthe distance between the dermo-epidermal boundary and the top of thepopulation of chromophores. This is in addition to the concentration anddepth of the chromophores. The problem was that the position of a spotwithin a three-dimensional CIE LMS color space was not necessarilyunique to a given set of measurements. The same position could beachieved by relative variation between two of the variables concerned.Previously, it had been necessary to make an estimate based on priorassumptions about the relationship between these variables. The analysisof a fourth or further wavelength band allows comparison with a notionalcolor space having four (or more) dimensions so that any position withinthat N dimensional space can be attributed to a unique depth,concentration and offset of a particular chromophore.

The present invention at least in its most preferred embodiments,enables the generation of information regarding a number of featuresregarding skin. To allow an accurate diagnosis of disorders of the skin,or the prognosis of treatment for such disorders, or the monitoring ofhealthy skin, it is important that the spatial relationship betweenthese features can be understood. To facilitate the spatial correlationof two images, one showing the appearance of the skin and the othershowing a particular feature or of two images showing differentfeatures, we have developed a technique whereby a third image isgenerated. Thus we also provide a method of and apparatus for showingboth images together with the proportion or intensity of each adjustedthrough the use of a control of some means and this allows spatialcorrelation of the input images. For example the two original imagesmight be supplied in overlapping relation to a monitor screen of a PC,and the two images be relatively faded in and faded out in order tochange from viewing one image to another.

The display first shows an image, which may or may not be magnified, ofthe lesion as it actually appears to the eye or a surface microscopyview or an image taken using cross polarized illumination or an imageshowing a particular feature. By selecting a particular feature such asblood or areas of melanin invasion into the dermis or melanin within theepidermis etc. the display can then be faded to show this feature as animage. The fading allows a progression, or mixing, between the two viewsand is a convenient means of allowing a spatial correlation to be madebetween the features and the lesion image.

The images may be images representing the presence of particularexisting features of the skin or one or more of them may be computergenerated images representing the predicted effects of a treatment suchas a laser irradiation treatment. For example, as mentioned above, it ispossible to generate a color representation of the expected result of alaser irradiation treatment, and it would be possible to generate onesuch image for each of a set of different irradiation intensities. Thiswould enable a comparison of the different courses of treatment andwould allow selection of an appropriate treatment, for example the onegiving the most cosmetically acceptable result.

The analysis afforded by the present invention is also of value in theselection of the wavelength or wavelengths of any light (infra-red,visible or ultra-violet) irradiation treatment that may be indicated.For example, a knowledge of the constituents of a lesion allows aselection of a wavelength of light radiation which will be most stronglyand preferentially absorbed by constituents of that lesion. Also, aknowledge of the existence and structure and composition of overlyingtissue (including any discontinuities which it might contain) allows themost favorable compromise to be reached between low absorption in theoverlying tissue and high absorption in the lesion to be destroyed, thusproviding the most effective treatment with the lowest radiation dosage.Thus a laser of an appropriate wavelength may be selected, and/or avariable wavelength laser may be tuned, or an appropriate filter set maybe used in conjunction with a source of non-coherent radiation.

Such a technique may be applied not only to the skin as described above,but also to other epithelial or layered tissue of the human or animalbody. Such tissues include the epithelium of the cervix, the lining ofthe mouth, epithelia of the respiratory and digestive tracts and theeye, including such specialized tissues as the sclera, cornea andretina, and epithelia of internal organs such as the liver and bladder.

The mathematical model described accounts for components such as blood,melanin and collagen, and is generally applicable to epithelial orlayered tissues. It is therefore possible to predict the coloration orspectral composition of the tissue containing different amounts of thesecomponents. Characteristics of the components can thus be determined byan examination of measured spectral properties in the manner described.

For example, the cervix is covered with a stratified squamous epitheliumin which the distribution of blood, collagen and melanin may bedetermined, and information relating to this is useful in the monitoringand diagnosis of the general state of health especially with regard tocervical cancer. A second example relates to the interior of the humaneye. This includes various specialized tissues such as the sclera,cornea and retina containing blood, collagen and melanin in a layeredstructure. Information on the distribution of these components is usefulin monitoring the health of the eye.

In each case, the technique and mathematical model described can beadapted to take account of particular or additional light absorbing orlight scattering components or present in the tissue examined.

According to one particular aspect of the invention, a method of mappingthe papillary surface of an area of the dermis is provided.

The invention thus provides a way of obtaining a map which indicates thecontours of the papillary surface of the dermis. In its simplest form,this map is simply a line such as may be seen on a suitably preparedsection of biopsied skin. However, such line may be derived withoutincision and accompanying risk of infection, and it may also be derivedand inspected very much more quickly and without discomfort to thepatient.

This and other aspects of the invention are based on a realization thatthe thickness of the papillary dermis may be obtained by utilizing theproperty of human skin to vary its remittance of red and infraredradiation with varying papillary dermis thickness. In general, there isa relationship between remittance and thickness. The fact that red orinfrared radiation is also absorbed by other materials within the skin,particularly melanin and blood, is a complicating factor, but the layerthickness may still be measured by obtaining two red or infrared images,each at a different wavelength. The chosen wavelengths are notimportant, but one should be further into the infrared (i.e. at longerwavelength) than the other. Suitable wavelength bands are, as before,800-1000 nm and 600-800 nm, in that readily available infrared films andfilters may be used. The brightness of points within the image obtainedat the longer wavelength is affected to a greater extent by variationsin the papillary dermis thickness. Conversely, the image obtained atshorter wavelength will be affected to a greater extent by othermaterials such as melanin and blood. (In fact when operatingsufficiently far into the infrared, say at 1100 nm, the effects ofmelanin and blood become negligible, and it is possible to derive thenecessary information using a single wavelength measurement. But thisgreatly increases the cost of the detection and monitoring equipment.)By predicting the brightnesses of points of differing papillary dermisthickness and amounts of epidermal melanin which absorb near-infraredradiation at the two different infrared wavelengths, a reference graph(FIG. 1) can be obtained which consists of lines of constant papillarydermis thickness, wherein Primary 1 is the measurement made at thelonger (800-1000 nm) wavelength and Primary 2 is the measurement made atthe shorter (600-800 nm) wavelength. The absorption of blood withinthese wavelengths is very small (a hundredth of its peak value forvisible wavelengths at 600-800 nm and even less for 800-1000 nm) and toa first approximation may be ignored. Thus, by comparing values obtainedat these wavelengths with this graph, it is possible to ascertain thepapillary dermis thickness. However it is within the scope of thepresent invention to measure brightness at such a long infra-redwavelength e.g. 1100 nm that the brightness would vary to such anegligible extent with melanin and blood content that it wouldeffectively depend solely on the papillary dermis thickness. In such acase only one set of brightness measurements would be required.

To calculate the look-up graph shown in FIG. 1 the spectral remittanceof light from human skin can be calculated given knowledge of thequantity and position of substances within it. Such calculations can beperformed using a variety of mathematical means including Monte Carlomodeling and the Kubelka-Munk theory generating a value for P_(n) where$\begin{matrix}{{P_{n}\left( {\rho,d_{m},v} \right)} = \frac{\int_{0}^{\infty}{{R\left( {\rho,v} \right)}{\theta\left( {\lambda,d_{m}} \right)}^{2}{S(\lambda)}{S_{P_{n}}(\lambda)}{\mathbb{d}\lambda}}}{\int_{0}^{\infty}{{S(\lambda)}{S_{P_{n}}(\lambda)}{\mathbb{d}\lambda}}}} & {{Equation}\quad 34}\end{matrix}$where P_(n) represents the calculated or measured ratio of remitted toincident light for a particular wavelength function or filter S_(Pn)(λ)and incident light S(λ). θ represents the light absorbed within theepidermis with d_(m) representing the quantity of epidermal melanin. Rrepresents the light remitted from the dermis with ρ representing thequantity of blood and υ the thickness of the papillary dermis. P_(n) canalso be obtained through measurements on real skin rather than bycalculation.

This analysis can be extended to a more general case $\begin{matrix}{\begin{matrix}{P_{n}\left( {\rho_{1},\rho_{2},\rho_{3},\ldots\quad,p_{n},} \right.} \\{d_{1},d_{2},d_{3},\ldots\quad,d_{n},} \\{\phi_{m1},\phi_{m2},\phi_{m3},\ldots\quad,\phi_{mn},} \\\left. {d_{m},v,\kappa} \right)\end{matrix} = \frac{\int_{0}^{\infty}{{R\left( {\rho_{1},\rho_{2},\rho_{3},\ldots\quad,p_{n},d_{1},d_{2},d_{3},\ldots\quad,d_{n},\phi_{m1},\phi_{m2},\phi_{m3},\ldots\quad,\phi_{mn},d_{m},v} \right)}{\theta\left( {\lambda,d_{m},\kappa} \right)}^{2}{S(\lambda)}{S_{P_{n}}(\lambda)}\quad{\mathbb{d}\lambda}}}{\int_{0}^{\infty}{{S(\lambda)}{S_{P_{n}}(\lambda)}\quad{\mathbb{d}\lambda}}}} & {{Equation}\quad 35}\end{matrix}$

Where κ represent the amount of keratin and ρ₁, ρ₂, ρ₃, . . . , ρ_(n),the quantity of blood within n planes within the dermis parallel withthe skin surface of thickness d₁, d₂, d₃, . . . , d_(n). Within theseplanes, φ_(m1), φ_(m2), φ_(m3), . . . , φ_(mn), represent the quantityof melanin within the dermis. As with the simple case P_(n) can also beobtained through measurements on real skin rather than by calculation.For a detailed discussion of this technique please refer to “Anon-invasive imaging system for assisting in the diagnosis of melanoma”University of Birmingham, Symon Cotton, 1998.

The above discussion relates to measurements of the thickness of thepapillary dermis alone. However, according to Histology, a text andatlas, second edition, Michael Ross and Lynn Romrell, published byWilliams & Wilkins, “The papillary layer consists of loose connectivetissue. It is located immediately under the epidermis and is separatedfrom it by the basal lamina. The papillary layer is a relatively thinlayer extending into (and, thus, also constituting) the dermal papillaeand ridges.” In contrast the junction between the papillary dermis andreticular dermis is relatively smooth or at least varying with awavelength very large in contrast to the undulations of the papillarydermis.

It is apparent from this as the thickness of the papillary dermis, ν,refers to a particular sampling point, or rather the average over asampling area, measurements taken at a variety of points returninformation on the thickness of the papillary dermis at these points.Further to this if it is assumed that the papillary dermis constitutesthe dermal papillae and also that the junction between the papillarydermis and reticular dermis is smooth, or at least varies on a scalemuch larger than the dermal papillae, measurements made from a series ofpoints ν₁, ν₂, ν₃, . . . , ν_(n), as shown in FIG. 8, will—if displayedspatially—show the undulations in the papillary dermis. Furthermeasurements can be performed on the height of a particular dermalpapilla by subtracting a local minimum, shown in FIG. 8 as min1 (ν₂),from a local maximum, shown in FIG. 8 as max1 (ν₁). Examples showingdermal papillae generated using this method are shown in FIGS. 12 and13.

As discussed further by Ross and Romrell “They [dermal papillae] arecomplemented by what appear to be a series of similar projections orevaginations, called epidermal ridges or rete ridges, which project intothe dermis.” It is clear from this that information regarding the reteridges can be obtained in a similar manner as the rete ridges and dermalpapillae fit together and are therefore the inverse of one another. Forinstance the depth of an individual peg being calculated from max1−min1.To generate a three dimensional representation or two dimensionalsegment showing a number of rete ridges requires a calculation, C−ν_(n),where C is a constant greater than any of the max1−max2 measurements.

It is apparent from this that measurements of the papillary dermisthickness, ν, measured over an area or along a line when suitablyinterpreted can impart information regarding the dermal papillae andrete ridges. In particular if the thickness of the papillary dermis ismeasured over an area or along a line and then shown graphically theundulations of the dermal papillae can be observed. As the rete ridgesextend down from the epidermis filling the void between the dermalpapillae it also becomes evident that the inverse of such a measure—suchas a constant value minus the papillary dermis thickness—givesinformation regarding the rete ridges.

An example of this is shown in FIG. 9 where the dermal papillaepertaining to an area of skin in the shoulder region are shown risingfrom the dermis. In conjunction with this the rete ridges can be seendescending.

In the most preferred embodiments of the invention, means is providedfor monitoring the intensity of the light remitted from a plurality oflines or a two-dimensional array of points, and preferably with aresolution of at least 20 lines or dots per mm.

This allows the production of an analogue of a three-dimensional imagewhich can be printed or displayed on a monitor screen, and in the lattercase, the use of suitable software will enable the image to be rotatedso that its appearance can be viewed from a plurality of differentangles.

A higher resolution may be obtained, and will indeed be necessary ifinspection of a highly magnified image of the remitted light is to beobtained, but our tests have shown that a very high resolution is notnecessary for many purposes. In a particularly preferred apparatus, animage of remitted light is captured using a digital camera in which useis made of a charge coupled device measuring 20×15 mm with a resolutionof 800×600 pixels.

Such an image may take the form of a series of lines each of whichfollows the contour of the mapped surface while remaining constant inone of three orthogonal axes. Alternatively, it may comprise lines ofequal contour, or it may be constituted as a continuous tone or coloredpicture of the papillary surface over the area being inspected.

It is implicit in what has been stated above that no account is taken ofany variations in the shape of the boundary between the papillary dermisand the reticular dermis at the intradermal junction. It is assumed thatthe intradermal junction is flat. In fact, as mentioned there arevariations in the thickness of the papillary dermis when the presence ofthose papillae is discounted, but those variations are of longwavelength in comparison with variations due to the papillae and theymay be neglected.

Inspection and analysis of the architecture of the dermal papillae andthe epidermal rete ridges at the dermo-epidermal junction allowsinformation to be derived which is of considerable importance toclinicians in order to assist them in diagnosing or assessing theprogress of a range of dermatological phenomena.

Examples include the blistering diseases Pemphigus vulgaris and bullouspemphigoid. While these diseases appear clinically similar, they havevery different prognoses and they require different management.Pemphigus vulgaris manifests itself as blisters within the thickness ofthe epidermis which do not distend the local dermo-epidermal boundaryarchitecture, and it is potentially fatal with a 10% mortality rate.Bullous pemphigoid, however, gives rise to sub-epidermal blisteringwhich does distend the local dermo-epidermal boundary architecture:prognosis is good, and the disease tends to subside over a number ofmonths.

The dermo-epidermal boundary architecture is important in thedifferentiation between benign and malignant melanoma, and inidentifying the presence of fibrosis within a melanoma. It is alsoimportant when assessing the extent of basal cell carcinomas andsquamous cell carcinomas.

The present invention includes any method of analyzing biological tissuecomprising illuminating the tissue with light, spectrally measuring andanalyzing the differences between the incident and remitted light, theanalysis of this data to define a parameter of the tissue, thenormalization of the data to a standard value of that parameter using apredictive mathematical model of the optical properties of thebiological tissue, and the subsequent measurement of a further parameterfrom that normalized data, preferably with more than one sequentialnormalization and analysis step to define further parameters.

The invention also extends to any method for analyzing biological tissuecomprising the illumination of the tissue with light, the spectralmeasurement of the differences between the incident and remitted light,the analysis of these data by comparison of features present withinthese data with a previous mapping of these features to components orstructures present in the tissue. In such a method, it is preferred thatthe previous mapping of features is achieved by measuring samples oftissue experimentally.

Alternatively or in addition, it is preferred that the mapping betweenfeatures of the data and components or structures in the tissue takingthe form of a multi dimensional table, with a dimension for eachmeasurable component or structure. A feature of the spectral data may beused to select a sub set of these tables, and one or more features aresubsequently used to select further sub sets of tables relating tocomponents or structures within the tissue.

The present invention will now be described in further detail and withreference to the accompanying drawings, in which:-

FIG. 1 is a graph showing variation of brightness with papillary dermisthickness for primaries 1 and 2, as described hereinabove;

FIG. 2 is a schematic cross-sectional view through a section of skinillustrating melanin descent into the papillary dermis;

FIG. 3 is a schematic cross-sectional view through a section of skinillustrating normal, healthy regions and an abnormal region where, inthis case, melanin descent into the papillary dermis and the reticulardermis has taken place;

FIG. 4 is a block diagram showing the steps involved in one embodimentof the method of the present invention;

FIG. 5 is a diagram showing the predicted surface of normal skincoloration within a three-dimensional color space;

FIG. 6 is a diagram showing coloration within the skin cancer that isshown in FIG. 7 in the same 3-D color space as depicted in FIG. 5,wherein areas of normal and abnormal coloration are shown;

FIG. 7 is a photographic image of the skin cancer;

FIG. 8 shows measurements of the dermal papillae and rete ridges, asdescribed hereinabove,

FIGS. 9 to 11 are diagrammatic representations of sections through humanskin such as may be revealed by conventional biopsy techniques,

FIGS. 12 and 13 are representations of the dermo-epidermal boundary suchas may be mapped by the present invention

FIG. 14 is a schematic diagram of apparatus according to this invention,and.

FIG. 15 shows representations of the rete ridges (top) and dermalpapillae(bottom) from an area of skin ascertained by using the techniqueof the present invention.

FIG. 16 shows a representation of a basal cell carcinoma ascertained byusing the technique of the present invention.

FIGS. 1, 2 and 8 have been mentioned above.

Referring now to FIG. 3 of the drawings, a schematic skin section isshown wherein melanin (indicated by the black circles in FIG. 3) innormal healthy skin are present in the lower part of epidermis 10adjacent but above the dermo-epidermal junction 12 between the epidermisand the papillary dermis 14. The Breslow thickness referred to above isthe depth of melanin invasion in millimeters measured from granularlayer 16 which is a layer in the epidermis 10 where the skin goes scalyand forms the tough outer cornified layer 18. In the abnormal region ofthe skin, the melanin is shown as having descended not only into thepapillary dermis 14, but also into the underlying reticular dermis 20lying above the subcutaneous fat layer 22. It is to be appreciated that,in other cases, melanin decent can be into any layer of the skin and mayeven be into the subcutaneous fat layer 22.

Referring now to FIG. 4, there is shown a block diagram illustrating thesteps involved in a typical method of measurement in accordance with thepresent invention. In FIG. 4, block 38 exemplifies method step (i) abovethe determination of papillary dermis thickness by shining infraredlight at two wavelengths on an area of skin being subjected tomeasurement and measuring the amount of light reflected from a pluralityof points within that area. Block 40 exemplifies method step (ii) abovethe acquisition of an image at visible wavelengths of the same skinarea. This can be by CCD camera, digitized film or any other convenientmeans. Block 42 exemplifies method step (iii) above the transformationof the image into corrected color space of the skin model at apredetermined papillary dermis thickness. Block 44 exemplifies methodsteps (iv and v) above—the identification of regions containing dermalmelanin, by comparing the corrected skin color co-ordinates with thereference color co-ordinate range. Block 46 exemplifies method step (vi)above—use of the corrected color space to calculate the amounts ofepidermal melanin within normal regions adjacent to the regionscontaining dermal melanin and use thereof to give an indication of theamounts thereof which exist in the regions containing dermal melanin.Block 48 exemplifies a first part of method step (vii) above—calculationof dermal invasion using the measured coloration of the abnormal regionsand the calculated amount of epidermal melanin from 46. Block 50exemplifies a second part of method step (vii) above—transformation ofthe calculated dermal invasion of melanin into either the Breslowthickness or the Clark's level of invasion. This can be reported aseither representing the maximum invasion or as an image showing invasionover the skin.

Referring now to FIG. 5, the shaded surface indicates the range ofcolorations which can exist in normal healthy skin corrected to thepredetermined papillary dermis thickness. Skin colorations which departfrom this surface are indicative of dermal melanin.

Referring now to FIGS. 6 and 7, it can be seen that a region of the skinwhich is shown in FIG. 7 and which is indicated by arrow H in FIG. 6lies at a position corresponding to part of the shaded surfaceillustrated in FIG. 5 and is indicative of normal healthy skin, whereasan adjacent region indicated by arrow U in FIG. 6 lies outside suchsurface and is indicative of skin containing dermal melanin. Comparisonof the coloration of these two adjacent regions H and U enables thedepth of melanin invasion in the abnormal region of the skin in FIG. 7to be computed.

FIG. 9 is an illustration of a section through normal healthy skinshowing the epidermis, the papillary dermis and the reticular dermis,and shows the irregular dermo-epidermal boundary formed between thepapillary dermis and the epidermis by the interpenetrating dermalpapillae and the rete ridges of the epidermis.

FIG. 10 is an illustration of a section through skin showing a blisterdue to bullous pemphigoid which gives rise to sub-epidermal blisteringwhich distends the local dermo-epidermal boundary architecture.

FIG. 11 is an illustration of a section through skin showing a blisterdue to Pemphigus vulgaris which is located within the thickness of theepidermis and which does not distend the local dermo-epidermal boundaryarchitecture.

FIGS. 12 and 13 are maps of the dermo-epidermal boundary provided by theadoption of one aspect of the present invention, each representing askin area of about 0.75 mm square.

In both cases the skin is normal. The shallow papillae and rete ridgesshown in FIG. 12 indicate that the skin is from an area which is notsubject to high stress in the day-to-day life of the subject. It is infact from the lower back. In FIG. 13, the dermo-epidermal boundary ismore sharply corrugated and with a shorter wavelength, indicating agreater stress to that area arising from the day-to-day life of thesubject. The FIG. 13 map is of skin from the shoulder. The greaterdegree of corrugation is associated with a greater need for elasticityand/or a greater need for a resistance to shear between the epidermisand the dermis.

Referring now to FIG. 14, a light source 1 is arranged to direct a beamof light onto a first filter wheel 2 which contains a number of holes 21to 26 each of which may selectively be brought into the light path. Onesuch hole is left empty for the direct transmission of light from thelight source 1, while the remainder contain screens, for example ofstainless steel wire gauze which serve as gray-scale filters, cuttingdown light-transmission without affecting its spectral characteristics.The number of gray-scale filters may be as high or as low as desired.Behind the first filter wheel 2 is a second filter wheel 3 whichaccommodates a number of color filters. Four such filters 31 to 34 areshown. Again, the number of color filters may be as high or as low asdesired. One such filter may be absent for the direct transmission oflight.

The color filters would together cover as much of the spectrum asrequired, for example from the infra red, through to the ultra violet.For the purpose of reliably measuring the concentration of collagenwithin the papillary dermis, it would be possible to operate at a singlewavelength of around 1050 nm, for example using a 10 nm full width-halfmaximum bandpass filter centered on that wavelength. This is because theabsorption of light of that wavelength by melanin is negligible.However, sensors which are capable of operating in that region areexpensive and it is preferred to use shorter wavelengths and to takemeasurements at two different wavelengths where the absorptioncharacteristics of melanin and blood are different so that melanin andblood concentrations can be calculated and/or compensated for. It is inparticular preferred to use two 10 nm full width half maximum bandpassfilters respectively centered on 694 nm and 940 nm. Other color filtersmay be used as desired for monitoring particular wavelengths orwavelength bands. A particularly preferred filter set includes five 10nm full width half maximum bandpass filters respectively centered on420, 568, 580, 694 and 940 nm, and three broad band (80 nm) filterscentered on 450, 550 and 650 nm.

The reason for using gray-scale filters is that a rather high intensitylight source is required for obtaining measurements in the infra-redregion due to the low transmission of color filters passing light ofsuch wavelengths. In fact we presently prefer to use a xenon lightsource rated at 300 Watt. Direct transmission of such light, ortransmission through for example a yellow filter could burn out a sensorsuitable for monitoring in the infra-red. The use of a suitably selectedset of gray-scale filters enables a single light source and a singlesensor to be used, and this simplifies the apparatus and keeps costsdown. A suitable set of gray-scale filters comprises those passing 50%,10% and 1% of incident light

The light is passed to a bundle of optical fibers 4 through which it istransmitted to the skin S of the patient, or even to an appropriatephotographic image of that skin, via a polarizing filter 41. Remittedlight is carried back through a second polarizing filter 51 and a secondbundle of optical fibers 5 to a photo-receptor unit 6. In otherembodiments, the optical fibers 4, 5 run along an endoscope appropriatefor the in vivo examination of internal epithelial tissue.

The projected light is preferably polarized, and the remitted light issuitably cross-polarized before monitoring. This is especially suitablefor monitoring the presence of chromophores beneath the epidermis. Sincelittle scattering of light takes place in the epidermis, anycross-polarized light which is detected must have been remitted from orvia the (papillary) dermis, and this allows surface effects and theeffects of the epidermis to be eliminated. A similar effect can beachieved without using cross-polarized illumination by coating thesurface of the skin with a transparent oil which removes directreflections at the skin surface.

The two polarizing filters 41, 51 are set so that their respectiveplanes of polarization are at right angles, to eliminate specularlyreflected light. The photo-receptor unit 6, which may simply measure theintensity of the remitted light where a series of color filters is usedas illustrated, emits a signal to a comparator 7 which may beconstituted as a suitably programmed PC.

As previously mentioned, the photo-receptor is suitably a CCD array, forexample a 20×15 mm array adapted to resolve 800×600 pixels.

The use of the bundles of optical fibers adds greatly to the convenienceof use of the apparatus since a relatively small unit at the end of aflexible lead may thereby be brought to the patient's skin S: thus thephysical posture of the subject during measurement is largely irrelevantand he or she may be made as comfortable as possible.

The comparator 7 is arranged to process the signals received whichrelate to the intensity of light remitted at the wavelengths 694 nm and940 nm, and to derive therefrom a signal proportional to theconcentration of collagen within the papillary dermis.

The comparator 7 is suitably arranged to supply the results for eachpixel monitored via a processor 8 to a display monitor 9 and/or to aprinter 10. The processor 8 is arranged to take the signal proportionalto the collagen concentration and to use that signal as a measure ofaltitude to generate a relief map for printing or display. The processor8 is suitably programmed to allow rotation of the display of the reliefmap. Examples of such relief maps which show the architecture of thedermo-epidermal boundary constitute FIGS. 4 and 5 of this specification.

The present invention at least in its most preferred embodiments,enables the generation of information regarding a number of features ofany skin being examined. To allow an accurate diagnosis of disorders ofthe skin, or the prognosis of treatment for such disorders, or themonitoring of healthy skin, it is important that the spatialrelationship between these features can be understood. Such anunderstanding of the dermo-epidermal boundary is greatly facilitated bypreferred embodiments of the present invention in which such a map isprovided. Such a map may be provided within seconds. Previously,examination by biopsy could reveal contours along a single line section,or more than one section if sufficient biopsy material was taken, but itwould be at least several hours and could well be several days beforethe results were available to the clinician.

The comparator 7 may also receive signals relating to the intensity oflight remitted in the red, yellow and blue regions of the spectrum, andof remitted white light. The comparator is arranged to assign a notionalposition in a color space according to co-ordinates represented by thesered, yellow and blue values and to note that position having regard tothe infra-red value. Instead of measurements over the three primarywavebands, other filters may be provided so that the visible spectrum issplit up into four or more wavebands. This establishes four or moreco-ordinates, and the comparator may thus assign a notional position ina color space having four or more dimensions. That position can beunique as representing the presence, depth, offset and concentration ofany one or more of a range of chromophores within the skin. Thecomparator is suitably arranged to supply these results to a displaymonitor 9 and/or to a printer 10, and it may be arranged to pass controlsignals to the power supply 11 of a medical laser 12 or other source ofradiation whether coherent or non-coherent.

The control signal may thus be used for controlling or operating one ormore of the following: a display device such as a display monitor, aprinter, or a medical laser or other treatment device or apparatus.

The monitor 9 may be and preferably is provided with a touch screenwhereby any of the various operational or programming steps may beinitiated.

In some preferred embodiments of the invention, a mask is provided tosurround the area of skin being illuminated and remit light back to thephotoreceptor 6. The incorporation of a standard reflector into such amask simplifies calibration of the apparatus.

Thus by making use of the invention it is possible to obtain imageswhich correspond to: (a) the visual appearance of the skin surface; (b)the architecture of the dermo-epidermal boundary; and (c) the presenceof any chromophore within the skin, including its depth andconcentration, and an indication of its nature.

To facilitate the spatial correlation of two or more of such images, forexample one showing the appearance of the skin and another showing aparticular feature, or of two images showing different features, we havedeveloped a technique whereby a further image is generated. Thus we alsoprovide a method of and apparatus for showing both images together withthe proportion or intensity of each adjusted through the use of acontrol of some means and this allows spatial correlation of the inputimages. For example the two original images might be supplied inoverlapping relation to a monitor screen of a PC, and the two images berelatively faded in and faded out in order to change from viewing oneimage to another. This allows correlation between the surface appearanceof skin and any underlying feature which might have given rise to thatappearance. It is of particular interest in the examination of anylesion in the skin.

The display first shows an image, which may or may not be magnified, ofthe lesion as it actually appears to the eye or a surface microscopyview or an image taken using cross polarized illumination or an imageshowing a particular feature. By selecting a particular feature such asblood or areas of melanin invasion into the dermis or melanin within theepidermis etc. the display can then be faded to show this feature as animage. The fading allows a progression, or mixing, between the two viewsand is a convenient means of allowing a spatial correlation to be madebetween the features and the lesion image.

The images may be images representing the presence of particularexisting features of the skin or one or more of them may be computergenerated images representing the predicted effects of a treatment suchas a laser irradiation treatment. For example, as mentioned above, it ispossible to generate a color representation of the expected result of alaser irradiation treatment, and it would be possible to generate onesuch image for each of a set of different irradiation intensities. Thiswould enable a comparison of the different courses of treatment andwould allow selection of an appropriate treatment, for example the onegiving the most cosmetically acceptable result.

The analysis afforded by the present invention is also of value in theselection of the wavelength or wavelengths of any light (infra-red,visible or ultra-violet) irradiation treatment that may be indicated.For example, a knowledge of the constituents of a lesion allows aselection of a wavelength of light radiation which will be most stronglyand preferentially absorbed by constituents of that lesion. Also, aknowledge of the existence and structure and composition of overlyingtissue (including any discontinuities which it might contain) allows themost favorable compromise to be reached between low absorption in theoverlying tissue and high absorption in the lesion to be destroyed, thusproviding the most effective treatment with the lowest radiation dosage.Thus a laser of an appropriate wavelength may be selected, and/or avariable wavelength laser may be tuned, or an appropriate filter set maybe used in conjunction with a source of non-coherent radiation.

FIG. 15 shows a map of rete ridges above corresponding dermal papillaederived using this invention.

As illustrated by FIG. 16, the dermo-epidermal boundary architecture isimportant inter alia for assessing the extent of basal cell carcinomas.FIG. 16 is a map of the dermo-epidermal boundary which includes a partaffected by such a carcinoma. The contrast between well developed anddistinct papillae of healthy skin to the left of the Figure and the areaof almost destroyed papillae at the upper right section of the Figure iswell marked and clearly shows the boundary of such a carcinoma. Theinformation imparted by such a map of the dermo-epidermal boundary isplainly of value in assisting diagnosis and in the planning of surgicalexcision boundaries.

1. Apparatus for monitoring the presence of individual chromophores in atissue sample, said apparatus comprising: a light source operable toilluminate an area of a tissue sample with different wavelengths oflight; a photo-receptor arranged to receive light re-emitted by an areaof a tissue sample illuminated by said light source; and a spectroscopicanalyzer operable to utilize the spectral characteristics of re-emittedlight received by said photo-receptor and the spectral characteristicsof light projected by said light source to identify the presence of saidone or more chromophores within the area of the illuminated issue samplefrom which re-emitted light is received by said photo-receptor, whereinsaid spectroscopic analyzer comprises: records of the spectralcharacteristics of light re-emitted by reference samples of tissue ofknown structure containing identified chromophores; said spectroscopicanalyzer being arranged to compare the spectral characteristics ofre-emitted light received by said photo-receptor with said records toidentify the record including spectral characteristics most closelymatching the characteristics of said re-emitted light received by saidphoto-receptor and to output as an identification of individualchromophores present in the area of the illuminated issue sample fromwhich re-emitted light is received by said photo-receptor anidentification of the chromophores associated with said spectralcharacteristics by said record.
 2. Apparatus in accordance with claim 1,wherein said records comprise records of the spectral characteristics oflight remitted by reference samples of epithelial or epithelial andsub-epithelial tissue.
 3. Apparatus in accordance with claim 2, whereinsaid records comprise records of the spectral characteristics of lightremitted by reference samples of skin.
 4. Apparatus in accordance withclaim 1, wherein said records comprise records of the spectralcharacteristics of light remitted by reference samples of normal healthytissue.
 5. Apparatus in accordance with claim 1, wherein said recordscomprise records of calculated estimated spectral characteristics oflight re-emitted by samples of tissue of known structure containingidentified chromophores.
 6. Apparatus in accordance with claim 1 whereinsaid light source is operable to selectively illuminate an area of atissue sample with different wavelengths of light.
 7. Apparatus inaccordance with claim 6, wherein said light source further comprises aset of filters for selective substitution into the tissue-incident lightpath in order to selectively illuminate an area of tissue with differentwavelengths of light.
 8. Apparatus in accordance with claim 1, whereinsaid light source further comprises a polarizing filter arranged topolarize the light which illuminates said area of tissue.
 9. Apparatusin accordance with claim 8, further comprising a cross-polarizing filterarranged to cross-polarize light remitted from said area of tissuebefore it is received by said photo-receptor.
 10. Apparatus inaccordance with claim 1, wherein said light source is operable toilluminate said area of tissue with light having a wavelength in excessof 600 mm.
 11. Apparatus in accordance with claim 1, wherein saidphoto-receptor is operable to detect re-emitted light having wavelengthsin the 800 to 1000 nm band and the 600 to 800 nm band.
 12. Apparatus inaccordance with claim 1, wherein said photo-receptor is operable toreceive light remitted from an array of points and said spectroscopicanalyzer is operable to identify the presence of one or more individualchromophores at each of said points in said array.
 13. Apparatus inaccordance with claim 12, wherein said photo-receptor is operable toreceive light remitted from points with a resolution of at least 20lines or dots per mm.
 14. Apparatus in accordance with claim 1 whereinsaid spectroscopic analyzer is operable to determine on the basis of thespectral characteristics of said received re-emitted light the depth ofpapillary dermis of a tissue sample being monitored and to identify thepresence of said one or more individual chromophores within the area ofthe illuminated issue sample from which re-emitted light is received bysaid photo-receptor utilizing the spectral characteristics of saidreceived re-emitted light and said determined depth of papillary dermis.15. Apparatus in accordance with claim 1 wherein said light source,photo-receptor and spectroscopic analyzer are together adapted togenerate a signal indicative of the presence of chromophores which issubstantially independent to the variation of spectral characteristicsof re-emitted light arising due to the presence of a predeterminedchromophore.
 16. Apparatus in accordance with claim 15 wherein saidlight source and photo-receptor are arranged to project and detect lightat wavelengths which are substantially unaffected by the presence of apredetermined chromophore.
 17. Apparatus in accordance with claim 15wherein said spectroscopic analyzer is arranged to process dataindicative of the spectral characteristics of re-emitted light receivedby said photo-receptor to generate signals which are indicative of thepresence of chromophores which are substantially independent of thevariation in spectral characteristics arising due to the presence of apredetermined chromophore.
 18. Apparatus in accordance with claim 16wherein said predetermined chromophore comprises a chromophores selectedfrom melanin, blood and tattoo pigments.
 17. Apparatus according toclaim 1, further comprising a light guide of which a least part isflexible provided for conducting light between said light source, saidtissue sample and said photo-receptor.
 18. Apparatus according to claim17, wherein said light guide comprises an endoscope.
 19. Apparatusaccording to claim 17, wherein said light guide terminates in a headadapted for placing against an area of skin.
 20. Apparatus according toclaim 1, wherein said light source is operable to vary the size of thearea of tissue from which re-emitted light is received.
 21. Apparatus inaccordance with claim 1, wherein said light source is operable toproject light which either has a wavelength sufficiently far into theinfra-red that its absorption by melanin and blood is negligible, orwhich has at least two wavelengths of which at least one is in excess of600 nm, wherein said spectroscopic analyzer is operable to process dataindicative of the spectral characteristics of re-emitted light receivedby said photo receiver to determine variations in spectralcharacteristics arising independently of variations due to the presenceof melanin and blood and to utilize said determined independentvariations in spectral characteristics to generate a signal indicativeof the concentration of collagen within the papillary dermis of a tissuesample being monitored.
 22. Apparatus in accordance with claim 1 furthercomprising a signal generator operable to generate control signals onthe basis of the determination of the presence of one or morechromophores in a tissue sample by said spectroscopic analyzer, whereinsaid control signals are operable to control an apparatus selected froma display monitor, a printer, a medical laser and a treatment device.23. Use of apparatus according to claim 1 for deriving data relating tothe presence, depth, and concentration of chromophores and creating anddisplaying a map thereof.
 24. Use of apparatus according to claim 1 forderiving data relating to the presence, depth, and concentration of anychromophore selected from the group consisting of: melanin, blood,hemoglobin, oxy-hemoglobin, bilirubin, tattoo pigments or dyestuffs,keratin, collagen and hair.
 25. Use of apparatus according claim 1 formapping the extent of a basal cell carcinoma.
 26. A method of monitoringthe presence of individual chromophores in a sample of tissue,comprising: storing records of the spectral characteristics of lightre-emitted by reference samples of tissue of known structure containingidentified chromophores; illuminating an area of a tissue sample withdifferent wavelengths of light using a light source; receiving lightre-emitted by the illuminated area of tissue at a photo-receptor;comparing the spectral characteristics of re-emitted light received bysaid photo-receptor with said records to identify the record includingspectral characteristics most closely matching the characteristics ofsaid re-emitted light received by said photo-receptor; and outputting asan identification of the individual chromophores present in the area ofthe illuminated tissue sample from which re-emitted light is received bysaid photo-receptor an identification of the chromophores associatedwith said spectral characteristics by said record.
 27. A methodaccording to claim 26, wherein said records comprise records of thespectral characteristics of light remitted by reference samples ofepithelial or epithelial and sub-epithelial tissue.
 28. A methodaccording to claim 27, wherein said records comprise records of thespectral characteristics of light remitted by reference samples of skin.29. A method according to claim 26, wherein said records compriserecords of the spectral characteristics of light remitted by referencesamples of normal healthy tissue.
 30. A method according to claim 26,wherein said records comprise records of calculated estimated spectralcharacteristics of light re-emitted by samples of tissue containingidentified chromophores.
 31. A method according to claim 26, furthercomprising the steps of: deriving data relating to the presence, depth,and concentration of chromophores; and creating and displaying a mapthereof.
 32. A method according to claim 26, comprising: deriving datarelating to the presence, depth, and concentration of any chromophoreselected from the group consisting of: melanin, blood, hemoglobin,oxy-hemoglobin, bilirubin, tattoo pigments or dyestuffs, keratin,collagen and hair.
 33. A method according to claim 26 which furthercomprising: passing projected light and/or the remitted light throughone or more filters and spectroscopically analyzing the remitted lightsuch that the result of the analysis is independent of the presence of apredetermined chromophore in the tissue.
 34. A method according to claim33 wherein said light source and photo-receptor are arranged to projectand detect light at wavelengths which are substantially unaffected bythe presence of said predetermined chromophore.
 35. A method accordingto claim 33 further comprising: processing data indicative of thespectral characteristics of re-emitted light received by saidphoto-receptor to generate signals which are indicative of the presenceof chromophores which are substantially independent of the variation inspectral characteristics arising due to the presence of saidpredetermined chromophores; and comparing said processed data with saidrecords to identify the record having the most closely correspondingspectral data.
 36. A method according to claim 33 wherein saidpredetermined chromophore comprises a chromophore selected from melanin,blood and tattoo pigments.
 37. A method according to claim 26, furthercomprising: determining the depth of papillary dermis of a tissue samplebeing monitored on the basis of the spectral characteristics of saidreceived re-emitted light; and identifying the presence of individualchromophores within the area of the illuminated issue sample from whichre-emitted light is received utilizing the spectral characteristics ofsaid received re-emitted light and said determined depth of papillarydermis.